Hung C H, Lee M C, Lin J Y
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, ROC.
FEBS Lett. 1994 Oct 24;353(3):312-4. doi: 10.1016/0014-5793(94)01066-8.
Native Acacia confusa trypsin inhibitor (ACTI) contains two disulphide bonds; one is an intrachain disulphide bond (Cys40-Cys86), located in the A-chain, while the other is an interchain disulphide bond (Cys133-Cys141) connecting the A- and B-chain; the inhibitor consists of 175 amino acid residues, 136 residues in the A-chain and 39 residues in the B-chain. The putative reactive site of ACTI is located at Lys64, while for all other Kunitz family trypsin inhibitors it is at Arg64. When the Lys64 residue of ACTI was converted into Ile or Arg by site-specific mutagenesis, the K64I mutant completely lost its inhibitory activity but the K64R mutant retained most of its inhibitory activity. The C133G mutant lost its inhibitory activity while the C40G mutant did not. This suggests that the interchain disulphide bond (Cys133-Cys141) linking two beta-strands of the six-strand beta-barrel is essential for ACTI inhibitory activity, while the intrachain disulphide bond (Cys40-Cys86) connecting the two loops is non-essential.
天然相思子胰蛋白酶抑制剂（ACTI）含有两个二硫键；一个是位于A链的链内二硫键（Cys40-Cys86），另一个是连接A链和B链的链间二硫键（Cys133-Cys141）；该抑制剂由175个氨基酸残基组成，A链有136个残基，B链有39个残基。ACTI的假定活性位点位于Lys64，而所有其他Kunitz家族胰蛋白酶抑制剂的活性位点位于Arg64。当通过定点诱变将ACTI的Lys64残基转化为Ile或Arg时，K64I突变体完全丧失其抑制活性，但K64R突变体保留了大部分抑制活性。C133G突变体丧失了其抑制活性，而C40G突变体则没有。这表明连接六链β桶的两条β链的链间二硫键（Cys133-Cys141）对ACTI的抑制活性至关重要，而连接两个环的链内二硫键（Cys40-Cys86）则是非必需的。
